Investigation of surface potential asymmetry in phospholipid vesicles by a spin label relaxation method.

In earlier work, Castle and Hubbell (1976) demonstrated the use of a spin-labeled amphiphile as a probe for the electrostatic potential at the outer surface of charged phospholipid vesicles. In recent experiments, we have shown that the hydrophobic anion tetraphenylboron (TPB) promotes transbilayer migration of the probe molecule. Relaxation data recorded following the rapid mixing of the probe with TPB-containing vesicle samples provides information about the electrostatic potentials at both the outer and inner vesicle surfaces. The measured potentials for both surfaces of asymmetrically screened vesicles were found to be in good agreement with theoretical values calculated using their known surface charge density. The method is also sensitive to transmembrane potentials as indicated by the response of the label to potentials created with the use of potassium concentration gradients and valinomycin.     

Inositol regulates phosphatidylglycerolphosphate synthase expression in Saccharomyces cerevisiae.

The enzyme phosphatidylglycerolphosphate synthase (PGPS; CDPdiacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase; EC 2.7.8.5) catalyzes the committed step in the synthesis of cardiolipin, a phospholipid found predominantly in the mitochondrial inner membrane. To determine whether PGPS is regulated by cross-pathway control, we analyzed PGPS expression under conditions in which the regulation of general phospholipid synthesis could be examined. The addition of inositol resulted in a three- to fivefold reduction in PGPS expression in wild-type cells in the presence or absence of exogenous choline. The reduction in enzyme activity in response to inositol was seen in minutes, suggesting that inactivation or degradation of the enzyme plays an important role in inositol-mediated repression of PGPS. In cho2 and opi3 mutants, which are blocked in phosphatidylcholine synthesis, inositol-mediated repression of PGPS did not occur unless choline was added to the media. Three previously identified genes that regulate general phospholipid synthesis, INO2, INO4, and OP11, did not affect PGPS expression. Thus, ino2 and ino4 mutants, which are unable to derepress biosynthetic enzymes involved in general phospholipid synthesis, expressed wild-type levels of PGPS activity under derepressing conditions. PGPS expression in the opi1 mutant, which exhibits constitutive synthesis of general phospholipid biosynthetic enzymes, was fully repressed in the presence of inositol and partially repressed even in the absence of inositol. These results demonstrate for the first time that an enzymatic step in cardiolipin synthesis is coordinately controlled with general phospholipid synthesis but that this control is not mediated by the same genetic regulatory circuit.     

Effect of liming on spore germination,germ tube growth and root colonization by vesicular-arbuscular mycorrhizal fungi

Summary The effect of soil acidity on spore germination, germ tube growth and root colonization of vesicular-arbuscular mycorrhizal (VAM) fungi was examined using a Florida Ultisol. Soil samples were treated with 0, 4, 8 and 12 meq Ca/MgCO₃/100 g soil and each lime level received 0, 240, and 720 ppm P as superphosphate. Corn (Zea mays L.) was planted in the soil treatments, inoculated with eitherGlomus mosseae orGigaspora margarita spores and grown for 31 days. Acid soil inhibits mycorrhizal formation byG. mosseae through its strong fungistatic effect against the spores. The dolomitic lime increased mycorrhizal formation by both fungal species.G. margarita is much less sensitive to acidic conditions thanG. mosseae. Al ions are a very important component of the fungistatic property against the VAM symbiosis. VAM fungus adaptation may be important for plants growing on infertile acid soils if soil inoculation with these fungi is to contribute significantly to low-input technology for tropical agricultural systems.     

Estimation of inter-residue distances in spin labeled proteins at physiological temperatures: experimental strategies and practical limitations.

Magnetic dipolar interactions between pairs of solvent-exposed nitroxide side chains separated by approximately one to four turns along an alpha-helix in T4 lysozyme are investigated. The interactions are analyzed both in frozen solution (rigid lattice conditions) and at room temperature as a function of solvent viscosity. At room temperature, a novel side chain with hindered internal motion is used, along with a more commonly employed nitroxide side chain. The results suggest that methods developed for rigid lattice conditions can be used to analyze dipolar interactions between nitroxides even in the presence of motion of the individual spins, provided the rotational correlation time of the interspin vector is sufficiently long. The distribution of distances observed for the various spin pairs is consistent with rotameric equilibria in the nitroxide side chain, as observed in crystal structures. The existence of such distance distributions places important constraints on the interpretation of internitroxide distances in terms of protein structure and structural changes.     

Response of recruitment to light availability across a tropical lowland rain forest community

1.  Many hypotheses about species coexistence involve differential resource use and trade-offs in species' life-history traits. Quantifying resource use across most species in diverse communities, although, has seldom been attempted.
2.  We use a hierarchical Bayesian approach to quantify the light dependence of recruitment in 263 woody species in a 50-ha long-term forest census plot in Panama. Data on sapling recruitment were obtained using the 1985–1990 and 1990–1995 census intervals. Available light was estimated for each recruit from yearly censuses of canopy density.
3.  We use a power function (linear log–log relationship) to model the light effect on recruitment. Different responses of recruitment to light are expressed by the light effect parameter b . The distribution of b had a central mode at 0.8, suggesting that recruitment of many species responds nearly linearly to increasing light.
4.  Nearly every species showed increases in recruitment with increasing light. Just nine species (3%) had recruitment declining with light, while 198 species (75%) showed increasing recruitment in both census intervals. Most of the increases in recruitment were decelerating, i.e. the increase was less at higher light ( b  < 1). In the remaining species, the response to light varied between census intervals (24 species) or species did not have recruits in both intervals (41 species).
5.   Synthesis. Nearly all species regenerate better in higher light, and recruitment responses to light are spread along a continuum ranging from modest increase with light to a rather strict requirement for high light. These results support the hypothesis that spatio-temporal variation in light availability may contribute to the diversity of tropical tree species by providing opportunities for niche differentiation with respect to light requirements for regeneration.     

Variations in Cellulosic Ultrastructure of Poplar

A key property involved in plant recalcitrance is cellulose crystallinity. In an attempt to establish the typical diversity in cellulose ultrastructure for poplar, the variation and distribution of supramolecular and ultrastructural features, including the fraction of crystalline cellulose forms $ {\text{I}}_{\alpha } $ and $ {\text{I}}_{\beta } $ , para-crystalline cellulose and amorphous cellulose content were characterized. In this study, the percent crystallinity (%Cr) and lateral fibril dimensions of cellulose isolated from poplar were determined for 18 poplar core samples collected in the northwestern region of the USA.     

Porphyrin-based Photocatalytic Nanolithography: A NEW FABRICATION TOOL FOR PROTEIN ARRAYS*

Nanoarray fabrication is a multidisciplinary endeavor encompassing materials science, chemical engineering, and biology. We formed nanoarrays via a new technique, porphyrin-based photocatalytic nanolithography. The nanoarrays, with controlled features as small as 200 nm, exhibited regularly ordered patterns and may be appropriate for (a) rapid and parallel proteomics screening of immobilized biomolecules, (b) protein-protein interactions, and/or (c) biophysical and molecular biology studies involving spatially dictated ligand placement. We demonstrated protein immobilization utilizing nanoarrays fabricated via photocatalytic nanolithography on silicon substrates where the immobilized proteins are surrounded by a non-fouling polymer background.Biomolecular arrays facilitate molecular aggregate investigation and high throughput analysis of immobilized biomolecules. Current biomolecular arraying capabilities are limited by relatively large sample volumes (typical spot sizes are on the order of ∼100–200 μm) and relatively long incubation times (1). Despite their limitations, protein microarray applications include autoantibody profiling, antibody response profiling, and identification and detection of bacterial and protein analytes as well as disease proteomics (oncoproteomics) (2–5). Typically a specific analyte is bound and detected by fluorescence, resulting in an expression profile or protein atlas.Nanoarrays are expected to expand the use of biomolecular arrays beyond drug discovery, medical diagnostics, and genetic testing to include point-of-care and in-the-field applications (6). We present a rapid and low cost photocatalytic lithography method for generating biomolecular nanoarrays on a non-fouling background appropriate for analysis of immobilized biomolecules.A number of publications have reviewed approaches to nanofabrication and bionanopatterning (7–9) of substrates including photolithography, contact printing (9, 10), imprint lithography (11–13), dip-pen lithography (14–17), and block co-polymer formation (18, 19). These approaches exhibit varying degrees of efficiency and success. Photocatalytic lithography (PCL)¹ is a more recently described technique that is capable of generating arrays. Lee and Sung (20) patterned silane layers by activating TiO₂ (a photocatalytic semiconductor) via 2-min exposure to UV light. They produced ∼600-nm parallel lines with 400-nm spaces, and they subsequently performed atomic layer deposition of zirconium dioxide (ZrO₂) onto exposed silanol groups (alternating octadecylsiloxane regions did not have necessary precursor molecules). The subsequent decomposition of alkylsiloxane monolayers with TiO₂ was reported to be 20 times faster than under UV irradiation in air.Previously we have described patterning via porphyrin-based PCL (21). Patterning is achieved within 10 s and with extremely low energy sources (an LED flashlight is sufficient). As presented below, we have miniaturized features patterned with this new technique and are now able to pattern on the scale of 200 nm; this is roughly half the reported scale achieved with PCL using photocatalytic semiconductors. We refer to patterning on this scale as porphyrin-based photocatalytic nanolithography (PCNL). We describe the implementation of porphyrin-based PCNL to form large scale nanoarrays appropriate for rapid, parallel, quantitative proteomics screening of immobilized biomolecules and to form spatially dictated ligand arrays for functional proteomics studies.     

Genetic evidence of frequent long-distance recruitment in a vertebrate-dispersed tree

The importance of dispersal for the maintenance of biodiversity, while long-recognized, has remained unresolved. We used molecular markers to measure effective dispersal in a natural population of the vertebrate-dispersed Neotropical tree, Simarouba amara (Simaroubaceae) by comparing the distances between maternal parents and their offspring and comparing gene movement via seed and pollen in the 50 ha plot of the Barro Colorado Island forest, Central Panama. In all cases (parent-pair, mother-offspring, father-offspring, sib-sib) distances between related pairs were significantly greater than distances to nearest possible neighbours within each category. Long-distance seedling establishment was frequent: 74% of assigned seedlings established > 100 m from the maternal parent [mean = 392 +/- 234.6 m (SD), range = 9.3-1000.5 m] and pollen-mediated gene flow was comparable to that of seed [mean = 345.0 +/- 157.7 m (SD), range 57.6-739.7 m]. For S. amara we found approximately a 10-fold difference between distances estimated by inverse modelling and mean seedling recruitment distances (39 m vs. 392 m). Our findings have important implications for future studies in forest demography and regeneration, with most seedlings establishing at distances far exceeding those demonstrated by negative density-dependent effects.